Method for producing L-amino acids by fermentation

ABSTRACT

The present invention provides an industrially efficient method for producing an L-amino acid useful as medicament, chemical agent, food material and feed additive, and the method comprising culturing in a medium a microorganism having an ability to produce the L-amino acid and having resistance to a DNA gyrase inhibitor or a microorganism having an ability to produce the L-amino acid and having both resistance to a DNA gyrase inhibitor and resistance to an aminoquinoline derivative, producing and accumulating the L-amino acid therein and recovering the L-amino acid therefrom.

BACKGROUND OF THE INVENTION

The present invention relates to a method for producing an L-amino acidby fermentation at high industrial efficiency.

As a direct fermentation method for producing and accumulating anL-amino acids directly from saccahride, there have been known methods inwhich mutant strains derived from wild-type strains of microorganismbelonging to the genus Corynebacterium, Brevibacterium, Escherichia,Serratia or Arthrobacter are employed For example, the following areknown as L-amino acid-producing mutants: auxotrophic mutants whichrequire amino acids, etc. (Japanese Published Examined PatentApplication No. 10037/1981), mutants which have resistance to amino acidanalogs and vitamins (Japanese Published Unexamined Patent ApplicationNos. 134993/1981 and 44193/1987), mutants which have both auxotrophicmutation and resistance mutation to amino acid analog(Japanese PublishedUnexamined Patent Application Nos. 31093/1975 and 134993/1981), mutantswhich have lowered degradability (Japanese Published Unexamined PatentApplication No. 273487/1988, and Japanese Published Examined PatentApplication No. 48195/1977), and mutants whose aminoacylt-RNA-synthesizing enzymes have a decreased substrate affinity (JapanesePublished Unexamined Patent Application No. 330275/1992).

It has also been known that the production of an amino acid can beimproved by using transformants obtained by transformation withrecombinant DNAs carrying genes involved in the biosynthesis of aminoacids (Japanese Published Unexamined Patent Application Nos. 893/1983,12995/1985, 210994/1985, 30693/1985, 195695/1986, 271981/1986, 458/1990and 42988/1990; Japanese Published Examined Patent Application s.42676/1989, 11960/1993 and 26467/1993).

For producing L-tryptophan, there has been a report that theproductivity of the amino acid was improved by giving resistance toaminoquinoline derivatives or to phenothiazine derivatives (JapanesePublished Unexamined Patent Application No. 112795/1992).

There have been a report that the expression of an operon involved inhistidine synthesis is increased in a DNA gyrase-deficient strain [Proc.Natl. Acad. Sci. USA, 84, 517 (1987)] and a report that the levels ofsome amino acid t-RNA species including His-tRNA are decreased in a DNAgyrase mutant strain [J. Mol. Biol., 66, 131 (1972)], however, no reporthas been made yet about the relation between resistance to DNA gyraseinhibitors and amino acid productivity.

SUMMARY OF THE INVENTION

An object of the present invention is to provide an industriallyefficient method for producing an L-amino acid useful as medicament,chemical agent, food material and feed additive.

The present invention relates to the following aspects (1) to (14).

(1) A method for producing an L-amino acid, which comprises:

(a) culturing in a medium a microorganism having an ability to producean L-amino acid and having resistance to a DNA gyrase inhibitor;

(b) producing and accumulating the L-amino acid in the culture; and

(c) recovering the L-amino acid from the culture.

(2) The method for producing an L-amino acid as described above in (1),wherein the DNA gyrase inhibitor is selected from the group consistingof nalidixic acid, oxolinic acid, coumermycin, novobiocin and the alkalimetal salts of these substances.

(3) The method for producing an L-amino acid as described above in (1),wherein the microorganism has resistance to an aminoquinolinederivative.

(4) The method for producing an L-amino acid as described above in (3),wherein the aminoquinoline derivative is selected from the groupconsisting of chloroquine, amodiaquine, pentaquine, primaquine and thealkali metal salts of these substances.

(5) The method for producing an L-amino acid as described above in anyone of (1) to (4), wherein the L-amino acid is L-histidine.

(6) The method for producing an L-amino acid as described above in (1)or (3), wherein the microorganism is selected from the group consistingof genera Serratia, Corynebacterium, Arthrobacter, Microbacterium,Bacillus and Escherichia.

(7) The method for producing an L-amino acid as described above in (6),wherein the microorganism is selected from the group consisting ofEscherichia coli H-9342 (FERM BP-6675) and Escherichia coli H-9343 (FERMBP-6676).

(8) A microorganism having an ability to produce an L-amino acid andhaving resistance to a DNA gyrase inhibitor.

(9) The microorganism described above in (8), wherein the DNA gyraseinhibitor is selected from the group consisting of nalidixic acid,oxolinic acid, coumermycin, novobiocin, and the alkali metal salts ofthese substances.

(10) The microorganism described above in (8) or (9), wherein themicroorganism has resistance to an aminoquinoline derivative.

(11) The microorganism described above in (10), wherein theaminoquinoline derivative is selected from the group consisting ofchloroquine, amodiaquine, pentaquine, primaquine, and the alkali metalsalts of these substances.

(12) The microorganism described above in (8), wherein the L-amino acidis L-histidine.

(13) The microorganism described above in any one of (8) to (12),wherein the microorganism is selected from the group consisting ofgenera Serratia, Corynebacterium, Arthrobacter, Microbacterium,Bacillus, and Escherichia.

(14) A microorganism selected from either Escherichia coli H-9342 (FERMBP-6675) or Escherichia coli H-9343 (FERM BP-6676).

DETAILED DESCRIPTION OF THE INVENTION

As the microorganism of the present invention, any microorganism can beused, so long as it has an ability to produce an L-amino acid and hasresistance to the DNA gyrase inhibitor. Additionally, it is preferablethat the microorganism has further resistance to an aminoquinolinederivative. Examples of the microorganism include microorganismsbelonging to the genus Serratia, Corynebacterium, Arthrobacter,Microbacterium, Bacillus, or Escherichia, such as Serratia ficaria,Serratia fonticola, Serratia liquiefaciens, Serratia marcescens,Corynebacterium glutamicum, Corynebacterium mycetoides, Corynebacteriumvariabilis, Corynebacterium ammoniagenes, Arthrobacter crystallopoietes,Arthrobacter duodecadis, Arthrobacter ramosus, Arthrobacter sulfureus,Arthrobacter aurescens, Arthrobacter citreus, Arthrobacter globiformis,Microbacterium ammoniaphilum, Bacillus subtilis, Bacillusamyloliquefacines and Escherichia coli.

As the DNA gyrase inhibitor for use in the present invention, anysubstance can be used, so long as it inhibits DNA gyrase, one of thetype II topoisomerases which are present in bacteria. For example,nalidixic acid, oxolinic acid, coumermycin and novobiocin can be used asthe DNA gyrase inhibitor. Additionally, the alkali metal salts of thesesubstances can be used as the DNA gyrase inhibitor. Herein, any alkalimetal such as sodium and potassium can be used as the alkali metals.

As the aminoquinoline derivative for use in the present invention, anysubstance can be used, so long as it has the aminoquinoline skeleton.For example, 4-aminoquinoline derivatives such as chloroquine andamodiaquine and 8-aminoquinoline derivatives such as pentaquine andprimaquine can be used as the aminoquinoline derivative. Additionally,the alkali metal salts of these substances can be used as theaminoquinoline derivative. All of these substances are known asantimalarial drugs. Herein, any alkali metal such as sodium andpotassium can be used as the alkali metals.

The microorganism of the present invention can be obtained by subjectinga microorganism having an ability to produce an L-amino acid to aconventional mutation treatment including ultraviolet irradiation andthe treatment with mutagen such as N-methyl-N′-nitro-N-nitrosoguanidine(NTG), culturing the resulting mutant strains under general conditionson an agar plate medium containing a DNA gyrase inhibitor at aconcentration at which the parent strain cannot grow or grow poorly, andselecting colonies which grow more rapidly than that of the parentstrain or colonies which are larger than the parent strain among theresulting colonies.

Further, the microorganism having both resistance to a DNA gyraseinhibitor and resistance to an aminoquinoline derivative can be obtainedby subjecting the DNA gyrase inhibitor-resistant strain to a mutationtreatment, culturing the resulting mutant strains on an agar platemedium containing an aminoquinoline derivative at a concentration atwhich the parent strain cannot grow or grows poorly, and selectingcolonies which are larger than the parent strain among the resultingcolonies.

As the microorganism having an ability to produce the amino acid, amicroorganism inherently having an ability to produce the amino acid canbe used; alternatively, a microorganism which is newly obtained bysubjecting a wild-type of a microorganism to produce the amino acid byknown methods can also be used.

The known methods include cell fusion method, transduction method, andother gene recombinant techniques [for all, see Molecular Cloning, ALaboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press(1989) (abbreviated as Molecular Cloning, 2nd ed. hereinbelow)], inaddition to the above mutation treatment.

The microorganism of the present invention can also be obtained, bypreparing a microorganism having resistance to a DNA gyrase inhibitor ora microorganism having both resistance to a DNA gyrase inhibitor andresistance to an aminoquinoline derivative by conventional mutationtreatment and then by subjecting the prepared mutant microorganism tothe above-described method to confer on the microorganism an ability toproduce an L-amino acid.

Specific examples of the microorganisms of the present invention includeEscherichia coli H-9342 (FERM BP-6675) and Escherichia coli H-9343 (FERMBP-6676).

The production of the L-amino acid by using the microorganism of thepresent invention can be carried out by an conventional method forculturing bacteria.

As the medium used for the production of L-amino acid, any of medium maybe used, so long as it appropriately contains a carbon source, anitrogen source, an inorganic substance and trace amounts of nutrientswhich the strain requires.

As the carbon source, carbohydrates such as glucose, fructose, lactose,molasses, cellulose hydrolysates, crude saccharide hydrolysates andstarch hydrolysates; organic acids such as pyruvic acid, acetic acid,fumaric acid, malic acid and lactic acid; and alcohol such as glycerinand ethanol can be used.

As the nitrogen source, ammonia; various inorganic salts such asammonium chloride, ammonium sulfate, ammonium acetate and ammoniumphosphate; ammonium salts of organic acids; amines; peptone, meatextract, corn steep liquor, casein hydrolysates, soybean cakehydrolysates, various fermented cells and digested matters thereof canbe used.

As the inorganic substance, potassium dihydrogen phosphate, dipotassiumhydrogen phosphate, magnesium phosphate, magnesium sulfate, magnesiumchloride, sodium chloride, ferrous sulfate, manganese sulfate, coppersulfate, calcium chloride and calcium carbonate can be used.

The microorganism is cultured under aerobic conditions such as shakingculture and aerated agitation culture, at a temperature within a rangeof 20 to 40° C., preferably within a range of 28 to 37° C. The pH of themedium is within a range of 5 to 9, preferably around neutrality. The pHof the medium is adjusted by using calcium carbonate, inorganic ororganic acids, alkali solutions, ammonia and pH buffers. Generally, theL-amino acid is produced and accumulated in the medium by culturing for1 to 7 days.

After completion of the culturing, the precipitates such as cells areremoved from the culture, and the L-amino acid can be recovered from theculture by means of ion exchange treatment method, concentration, etc.,in combination.

In accordance with the present invention, any L-amino acid can beproduced with no specific limitation, but includes for exampleL-histidine.

The present invention is further illustrated by the following Examples,which are not to be construed to limit the scope of the presentinvention.

EXAMPLE 1

Preparation of an L-histidine-producing Mutant Strain Having Resistanceto a DNA Gyrase Inhibitor or an L-histidine-producing Mutant StrainHaving Both Resistance to a DNA Gyrase Inhibitor and Resistance to anAminoquinoline Derivative

The L-histidine-producing mutant strain H-9340 having resistance to1,2,4-triazole alanine, which was derived from methionine-requiringEscherichia coli ATCC 21318 was subjected to a mutation treatment withN-methyl-N′-nitro-N-nitrosoguanidine (NTG) (0.2 mg/ml, 30° C., 30minutes) according to a conventional method and spread on a 1 g/liternoboviocin monosodium salt-containing agar plate medium [0.2% glucose,0.3% potassium dihydrogen phosphate, 0.6% disodium hydrogen phosphate,0.01% magnesium sulfate, 0.05% sodium chloride, 0.1% ammonium chloride,50 mg/liter required nutrient (DL-methionine) and 1.5% agar, pH 7.2].

The mutant strain was cultured on the agar plate medium at 30° C. for 2to 6 days, and the growing large colonies were picked up and separatedto obtain the strain H-9342.

Furthermore, the obtained colony was subjected to a mutation treatmentwith NTG (0.2 mg/ml, 30° C., 30 minutes), followed by spreading on anagar plate culture medium containing 150 mg/liter primaquine disodiumsalt. Culturing was carried out thereon at 30° C. for 2 to 6 days, andgrowing large colonies were picked up and separated to obtain the strainH-9343. The strains H-9340, H-9342 and H-9343 were deposited on Mar. 9,1999 with the National Institute of Bioscience and Human-Technology,Agency of Industrial Science and Technology (1-3, Higashi 1-chome,Tsukuba-shi, Ibaraki-ken, Japan) under the Budapest Treaty withaccession Nos. FERM BP-6673, FERM BP-6675 and FERM BP-6676,respectively.

EXAMPLE 2

Comparative Test of Growth on Agar Plate Culture Medium ContainingPrimaquine or Novobiocin

The growth of the mutant strains H-9342 and H-9343 obtained in Example 1was compared with the growth of the parent strain H-9340 on an agarplate medium containing primaquine or novobiocin.

Each of the mutant strains, which had been cultured in a natural mediumfor 24 hours and suspended in physiological saline, was spread at a celldensity of 1 to 10 cells/cm² on the agar plate medium containingprimaquine disodium salt or novobiocin monosodium salt at the sameconcentration as that at the time of acquisition of each mutant strain,and cultured at 33° C. for 4 days.

Growth or non-growth of each strains on the above media is shown inTable 1.

The parent strain H-9340 did not grow on any agar plate culture mediumcontaining either one of the chemical agents. Additionally, H-9342 didnot grow on the primaquine-containing culture medium.

TABLE 1 Additives for agar culture medium Bacterial PrimaquineNovobiocin strain No addition disodium salt monosodium salt H-9340 + − −H-9342 + − + H-9343 + + +

EXAMPLE 3

Production of L-histidine

The production of L-histidine using the mutant strains H-9342 and H-9343obtained in Example 1 and the parent strain H-9340 was carried out inthe following manner.

Each of the strains H-9340, H-9342 and H-9343 was inoculated in 6 ml ofa seed medium (2% glucose, 0.5% molasses, 1% corn steep liquor, 1.2%ammonium sulfate, 0.3% potassium dihydrogen phosphate, 0.015% magnesiumsulfate, 600 mg/liter DL-methionine, 100 mg/liter adenine, 3% calciumcarbonate, pH 6.2) in a large test tube, and cultured with shaking at30° C. for 12 hours.

Each of the resulting seed cultures (0.1 ml) was inoculated in 5 ml of aproduction medium (6% glucose, 1% corn steep liquor, 2.4% ammoniumsulfate, 0.4% potassium dihydrogen phosphate, 0.015% magnesium sulfate,10 mg/liter thiamine chloride salt, 10 mg/liter calcium pantothenate, 3%calcium carbonate, pH 6.5) in a large test tube and was then culturedwith shaking at 30° C. for 48 hours.

After culturing, the amount of L-histidine accumulated in the culturewas assayed by high-performance liquid chromatography.

The results are shown in Table 2.

Compared with the L-histidine productivity of the parent strain H-9340,the L-histidine productivity of the mutant strain H-9342 was improved;and compared with the L-histidine productivity of the mutant strainH-9342, the L-histidine productivity of the mutant strain H-9343 wasimproved.

TABLE 2 Bacterial strains L-histidine (g/l) H-9340 13.0 H-9342 15.7H-9343 16.5

Furthermore, 100 ml of the seed culture of H-9343 was inoculated in 600ml of a fermentation culture medium (6% glucose, 1% corn steep liquor,0.5% ammonium sulfate, 0.4% potassium dihydrogen phosphate, 0.05%magnesium sulfate, 100 mg/liter calcium chloride, pH 6.5) in a 2-litersmall fermentor, and the culturing was conducted at 30° C., at the rateof 800 rpm at an aeration volume of 1 liter/min. The pH adjustment andnitrogen source supply during culturing were carried out by usingaqueous ammonia, to maintain the pH at 6.5±0.2. Under appropriate supplyof glucose, ammonium sulfate and potassium dihydrogen phosphate, theculturing was conducted for 70 hours.

Consequently, the amount of L-histidine accumulated in the culture was46.5 g/liter. On the other hand, the amount of L-histidine accumulatedduring the culturing of H-9340 in the same manner was 27.7 g/liter.

In accordance with the present invention, a microorganism having anability to produce an L-amino acid and having resistance to a DNA gyraseinhibitor or a microorganism having an ability to produce an L-aminoacid and having both resistance to a DNA gyrase inhibitor and resistanceto an aminoquinoline derivative can be obtained and cultured in amedium, whereby the productivity of the L-amino acid can be improved sothat the L-amino acid can be industrially efficiently produced at lowcost.

What is claimed is:
 1. A method for producing L-histidine, whichcomprises: (a) subjecting a microorganism belonging to the genusEscherichia and having an ability to produce an L-amino acid as a parentstrain to a mutation treatment selected from ultraviolet irradiation andtreatment with mutagen to obtain mutant strains; (b) culturing theresulting mutant strains on an agar plate medium containing a DNA gyraseinhibitor at a concentration at which the parent strain cannot grow orat which the parent strain grows poorly and whereby mutant strains thathave a resistance to the gyrase inhibitor produce colonies that growmore rapidly than the parent strain or colonies that are larger thanthat of the parent strain; (c) selecting colonies that grow more rapidlythan the parent strain or colonies that are larger than that of theparent strain from among colonies on the agar plate medium to obtain amicroorganism belonging to the genus Escherichia having an ability toproduce L-histidine and having resistance to a DNA gyrase inhibitor; (d)culturing in a culture medium said microorganism; (e) producing andaccumulating L-histidine in the culture medium; and (f) recoveringL-histidine from the culture medium.
 2. The method for producingL-histidine according to claim 1, wherein the DNA gyrase inhibitor isselected from the group consisting of nalidixic acid, oxolinic acid,coumermycin, novabiocin and the alkali metal salts of these substances.3. The method for producing L-histidine according to claim 1, wherein,before step (d), the colonies selected by carrying out steps (b) and (c)are further selected for resistance to an aminoquinoline derivative bythe additional steps of culturing the colonies selected in step (c) onan agar plate medium containing an aminoquinoline derivative at aconcentration at which the parent strain cannot grow or at which theparent strain grows poorly and whereby mutant strains that have aresistance to the aminoquinoline derivative produce colonies that growmore rapidly than the parent strain or colonies that are larger thanthat of the parent strain and selecting colonies that grow more rapidlythan the parent strain or colonies that are larger than that of theparent strain among the resulting colonies to obtain a microorganismbelonging to the genus Escherichia having an ability to produceL-histidine and having resistance to DNA gyrase inhibitor and to anaminoquinoline derivative.
 4. The method for producing an L-histidineaccording to claim 3, wherein the aminoquinoline derivative is selectedfrom the group consisting of chloroquine, amodiaquine, pentaquine,primaquine and the alkali metal salts of these substances.
 5. A methodfor producing L-histidine, comprising the steps of: (a) culturing in aculture medium a microorganism having an ability to produce an L-aminoacid and having resistance to a DNA gyrate inhibitor, wherein themicroorganism is selected from the group consisting of Escherichia coliH-9342 (FERM BP-6675) and Escherichia coli H-9343 (FERM BP-6676); (b)producing and accumulating L-histidine in the culture medium; and (c)recovering the L-histidine from the culture medium.